Antibody therapy for use in the digestive tract

ABSTRACT

In accordance with the invention, the development and use of antibodies within the digestive tract is provided. Antibodies are described that are used to treat disorders associated with altered permeability of the digestive tract. Antibodies are described with increased stability within the environment of the digestive tract. Antibodies are described with enhanced permeability to a compromised digestive tract.

RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 13/452,026, filed Apr. 20, 2012, which is a divisional applicationof U.S. application Ser. No. 12/753,438, filed Apr. 2, 2010, now U.S.Pat. No. 8,182,818, issued May 22, 2012, which is a continuation ofInternational Application No. PCT/US2008/078543, which designated theUnited States and was filed on Oct. 2, 2008, published in English, whichclaims the benefit of U.S. Provisional Application Nos. 60/976,876 filedon Oct. 2, 2007; 61/015,507 filed on Dec. 20, 2007; and 61/055,215 filedon May 22, 2008. The entire teachings of the above applications areincorporated herein by reference.

FIELD OF THE INVENTION

This invention describes the development and use of antibodies withinthe digestive tract.

BACKGROUND OF THE INVENTION

Diseases and disorders of the digestive tract cause significantmorbidity and mortality and there is a need for new therapeutics andtherapeutic strategies. Diseases of the digestive tract includemucositis, aphthous stomatitis, esophagitis, inflammatory bowel disease,irritable bowel syndrome, celiac disease, trauma to the digestive tract,infections of the digestive tract and cancers of the digestive tract.

For some digestive tract diseases, there are no effective treatmentsavailable. One example of such an unmet medical need is mucositis, aserious and painful condition that results from radiation therapy and/orchemotherapy for cancer treatment. According to a recent report by theNational Comprehensive Cancer Network, mucositis is the most significantadverse symptom of cancer therapy reported by patients {Bensinger etal., 2008, J Natl Compr Canc Netw, 6 Suppl 1, S1-21 quiz 522-4}. Damagecan occur throughout the digestive tract and frequently results incessation or dose reduction of the cancer therapy {Blijlevens and Sonis,2007, Ann Oncol, 18, 817-26}. Oral mucositis presents with pain,erythema and deep, diffuse ulcers that can cause difficulty speaking,eating and swallowing and significantly impair daily functioning. Opioidanalgesia, IV hydration, use of a liquid diet and total parenteralnutrition may be used in patients suffering from oral mucositis.Intestinal mucositis presents with nausea, vomiting, abdominal pain anddiarrhea, sometimes with blood loss. It most commonly affects the smallintestine, but is also seen in the stomach and large intestine. There isa single medication approved for the treatment of mucositis, palifermin{Blijlevens and Sonis, 2007, Ann Oncol, 18, 817-26}, but it is only usedin a limited subset of patients. Therefore, there is a need foradditional therapeutics for mucositis.

Another example of an unmet medical need is recurrent aphthousstomatitis (RAS), a common oral disease, affecting 5-20% of the normalpopulation {Porter et al., 1998, Crit Rev Oral Biol Med, 9, 306-21}. RASpresents with recurrent bouts of rounded, shallow painful ulcers on themucosa of the mouth. The most common form is minor RAS, where the ulcersare usually less than 5 mm in diameter. The ulcers usually occur on thelabial and buccal mucosa and the floor of the mouth and are uncommon onthe gingival, palate or dorsum of the tongue. The lesions heal within10-14 days. Major RAS is less common, with larger ulcers that persistfor up to 6 weeks and often heal with scarring. The etiology of RAS isunknown, and there are no approved pharmacotherapies.

For some diseases of the digestive tract, treatments are alreadyavailable. For example, both small molecule and biological therapies areavailable for the treatment of Crohn's disease and ulcerative colitis,the two forms of inflammatory bowel disease {Kozuch and Hanauer, 2008,World J Gastroenterol, 14, 354-77}. However, all of these medicationshave limitations, either in efficacy or in safety, and there is a needfor new therapeutic approaches and strategies.

Antibodies are an important class of pharmaceuticals. Specific antibodytherapeutics have been shown to be highly effective in treating cancersand autoimmune disease, and their use has been of great benefit toafflicted patients. Antibodies are generally highly specific for aparticular target and thus tend to have less off-target toxicity than isseen with small molecule therapeutics.

It would be advantageous to be able to apply antibody therapeutics todiseases of the digestive tract. Most antibody therapies in current useare designed to be delivered systemically and are administered topatients by injection. Injected antibodies have been shown to be usefulin the treatment of inflammatory bowel disease, and may also be usefulin the treatment of other diseases of the digestive tract. However,administration of antibodies systemically may affect physiologicalprocesses throughout the body, rather than just within the digestivetract, and this may be disadvantageous for some diseases. For instance,anti-TNF antibodies used for the treatment of inflammatory bowel diseaseare associated with serious side effects {Lin et al., 2008, ClinImmunol, 126, 13-30}. Therefore, it would be useful to be able to applyantibody therapeutics directly to the digestive tract.

There have been two obstacles to the use of antibody therapeutics withinthe digestive tract. First, the digestive tract is a hostile environmentfor protein therapeutics, with low pH environments designed to denatureingested proteins and proteases designed to digest ingested proteins.The oral cavity contains proteases derived from the host and fromresident microorganisms. Polymorphonuclear leukocytes are secreted inthe crevicular fluid, and some of these cells lyse before they areswallowed, releasing lysozyme into the fluid of the oral cavity.Bacteria present as part of the normal oral flora express proteasescapable of degrading immunoglobulin. These proteases include IgA1protease, IdeS, argingipain and SpeB. The stomach is highly acidic, andcontains the protease pepsin, which is active at low pH. The smallintestine contains additional proteases, including trypsin andchymotrypsin. The bacteria of the large intestine further express variedproteases that degrade ingested protein. Therefore, there is a need todevelop approaches to deliver antibody therapeutics to the digestivetract where the antibody will not be degraded. This invention describesmethods and compositions for antibodies that have improved stabilitywithin the oral cavity and the gastrointestinal tract.

Second, many of the targets that would be useful for antibodytherapeutics have not been thought to be accessible to antibody appliedto the luminal surface of the digestive tract. One of the functions ofthe digestive tract is to create a barrier to prevent the entry into thesystemic circulation of a variety of foreign agents that enter thedigestive tract, including food and microorganisms. Under normalcircumstances, this barrier would exclude topically applied antibodyfrom accessing targets expressed on the basolateral face of theepithelial cells lining the digestive tract, or on the mucosa orsubmucosa. Therefore, there is a need to develop approaches to targetingantibodies to these targets that lie below the normal barrier of thedigestive tract. This invention describes the use of antibodies to treatpatients with altered permeability barriers in the digestive tract,allowing topically applied antibody to access the appropriate targets.

SUMMARY OF THE INVENTION

This invention describes the development and use of antibodies withinthe digestive tract. Antibodies are described that are used to treatdisorders associated with altered permeability of the digestive tract.Antibodies are described with increased stability within the environmentof the digestive tract. Antibodies are described with enhancedpermeability to a compromised digestive tract.

DEFINITIONS

For the purposes of the invention, the digestive tract consists of themouth, pharynx, esophagus, stomach, small intestine (duodenum, jejunum,ileum), large intestine (cecum, colon, rectum) and anus.

For the purposes of the invention, the “oral cavity” is understood toinclude the mouth, the pharynx and the esophagus. The term “oraldegradation” of an antibody is used herein to mean degradation of anantibody in the oral cavity by endogenous or exogenous enzymes presentin the oral cavity.

For the purposes of the invention, the “gastrointestinal tract”, or “GItract” is understood to include the stomach, small intestine (duodenum,jejunum, ileum), large intestine (cecum, colon, rectum) and anus. Theterm “gastric digestion” as used herein is understood to describedigestion in the stomach, small intestine and large intestine. The term“gastric degradation” of an antibody is used herein to refer todegradation of an antibody in the stomach, small intestine, largeintestine by endogenous or exogenous enzymes present in the stomach,small intestine and large intestine or due to exposure to acidicconditions during gastric digestion.

The terms “antibody” and “antibodies” are used herein to refer tocompositions or preparations comprising one or more antibodies. The useof the singular terms “a” or “an” or “the” antibody are not meant to belimited to a single antibody when it is clear that more than oneantibody is present in the composition or preparation. In addition,unless indicated otherwise, the singular term for “antibody” may includea collection of antibodies that are not necessarily heterogenous intheir structures or specificities.

The term “stabilized antibody” as used herein is understood to describean antibody or antibody preparation that has been processed to make itmore stable to degradation in the digestive tract when administeredtopically. A stabilized antibody excludes the addition of J chainproteins, secretory component, or other similar proteins that are usedby the secretory immune system to stabilize secreted antibody, whetherthose additional proteins are natural or synthetic. As compared to anantibody that has not been processed in accordance with the invention, astabilized antibody that is processed in accordance with the inventionis degraded more slowly or to a lesser extent by endogenous enzymespresent in the oral cavity or by exogenous enzymes derived frommicroorganisms resident in the oral cavity as compared to antibodies andantibody preparations that have not been treated or processed inaccordance with the invention. Alternatively or in addition, as comparedto an antibody that has not been processed in accordance with theinvention, a stabilized antibody that is processed in accordance withthe invention is degraded more slowly or to a lesser extent by gastricdigestion which includes digestion by endogenous or exogenous enzymespresent in the stomach, small intestine and large intestine and/or bythe acidic conditions present in the stomach. “Stabilized antibodies”are also referred to as “antibodies with enhanced stability todegradation in the oral cavity and/or the GI tract”.

The term antibody with enhanced mucosal permeability as used herein isunderstood to describe an antibody that has been processed to make itmore permeable to a compromised mucosal barrier, as compared to anantibody that has not been processed in accordance with the invention.

The term “targets below the mucosal barrier” is defined as targetslocated on the basal side of the epithelium, targets expressed in thesubmucosa, targets expressed in the lateral intercellular space, targetsexpressed in the lamina propria, targets expressed in the centralnervous system, and targets expressed in the systemic circulation.

The term “topical application” to the GI tract is defined as localand/or surface administration to the oral cavity, delivery by oral orrectal administration to the GI tract, or administration by any otherroute that brings the antibody in contact with the luminal aspect of theGI tract.

The term “apical receptors” refers to endogenous transmembrane proteinsexpressed in the cell membrane of cells facing the luminal side of theintestinal tract.

The term “TNF” as used herein is used to describe the cytokine TNF-α.

The term “autoimmune disease that targets the GI tract” is used todescribe those autoimmune diseases that are known to involve substantialdamage to the GI tract and consist of ulcerative colitis and Crohn'sdisease (together known as inflammatory bowel disease), type I diabetesmellitus and systemic lupus erythematosus (SLE). This definitionexcludes celiac disease.

The term “glycoform” is used herein to describe an antibody with adefined pattern of glycosylation. The pattern of glycosylation includesboth the location and structure of attached carbohydrate. A glycoformmay be defined by determination of the molecular structure.Alternatively, a glycoform may be defined by a pattern of lectinbinding.

The term “carbohydrate” in this application is used interchangeably withthe terms “glycan” or “oligosaccharide”.

As used herein the term “compromised” as it relates to the GI tract andthe mucosal barrier of the GI tract is understood to mean that one ormore areas of the GI tract is permeable such that an antibody applied tosuch permeable area of the GI tract is capable of crossing the mucosalmembrane of the permeable area of the GI tract while remaining intact.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to the application of antibodies and stabilizedantibodies to the gastrointestinal tract of a patient including apatient with altered permeability of the digestive tract.

This invention describes the use of topical antibody therapeutics foruse in patients with barrier defects of the digestive tract. In thesepatients, topically applied antibody is able to cross the mucosalbarrier and access targets below the mucosal barrier.

This invention also describes the development and use of antibodies withincreased stability in the digestive tract, also referred to herein asstabilized antibodies. Such antibodies of the invention may be used inpatients with barrier defects of the digestive tract or in patientswithout barrier defects of the digestive tract.

Stabilized Antibodies

Topical administration of antibody to the digestive tract is challengingbecause the digestive tract degrades and digests the topically appliedantibodies. Enzymes in the oral cavity, primarily derived from commensaland pathogenic bacteria living within the oral cavity, degrade antibodyin the oral cavity. In the stomach, the low pH and the protease pepsindegrade ingested immunoglobulin. In the small intestine, the enzymestrypsin and chymotrypsin, among others, degrade ingested antibody. Inthe large intestine, bacterially-derived proteases degrade ingestedantibody. Antibodies with improved stability in the oral cavity would bepreferred for topical application to the oral cavity. Antibodies withimproved stability to gastric digestion would be preferred for topicalapplication to the GI tract.

Immunoglobulins contain substantial amounts of carbohydrate. The hingeregion of IgG, IgA and IgD can contain both N- and O-linkedcarbohydrate, while other regions of the heavy chain contain primarilyN-linked carbohydrates. There is significant heterogeneity inimmunoglobulin glycosylation. For example, human IgG has a principalN-linked glycosylation site at Asn-297. There are 32 differentcarbohydrates that may be present at the Asn-297 site: 16% of sites arecomprised of a bi-antennary carbohydrates with both arms terminating ingalactose; in 35% of sites, a terminal galactose is missing from one ofthe arms, exposing a GlcNAc residue; in 35% of sites, the terminalgalactose is missing from both of the arms; the remaining 14% of sitescontain sialylated variants. Additional diversity arises from thepresence or absence of bisecting GlNAc residues and core fucose. Similarcomplexity and diversity is seen with other isotypes and in otherspecies.

Carbohydrates on immunoglobulin serve many functions. They participatein binding to Fc receptors, facilitate cellular transport, secretion andclearance, bind to pathogens, maintain solubility and conformation, andparticipate in binding events to lectins such as mannan-binding lectin.In addition, carbohydrate plays a role in protecting immunoglobulin fromproteolytic degradation.

The hinge region is the site on the immunoglobulin molecule that is mostsensitive to proteolysis. Human IgA1 contains a 23 amino acid hingeregion with 9 potential glycosylation sites, of which five (Thr-228,Ser-230, Ser-232, Thr-225, and Thr-236) are fully or partially occupied;a sixth sugar is present at Ser-224, Thr-233 or Ser-240 on 5-10% of IgA1molecules. Bovine IgG1 has an extended hinge region that resembles thehinge region of human and mouse IgA1. Bovine IgG1 also contains Ser andThr residues in the hinge region and appears to contain O-linkedcarbohydrates, based on binding of the lectin jacalin {Porto et al.,2007, J Dairy Sci, 90, 955-62}. Therefore, some of the stability ofbovine immunoglobulin to proteolytic digestion appears to be due toO-linked glycosylation at the hinge region. Some mouse IgG2b moleculesare O-glycosylated at Thr-221A in the hinge region, predominantly with atetrasaccharide composed of Gal-NAc, galactose, and twoN-glycosylneuraminic acid residues. The carbohydrate addition covers theportion of the hinge region that is cleaved by papain and results inincreased resistance to papain digestion {Kim et al., 1994, J Biol Chem,269, 12345-50}.

Glycosylation outside of the hinge region is primarily N-linkedglycosylation. Glycosylation of immunoglobulin outside of the hingeregion can also affect the sensitivity to proteolysis. U.S. Pat. No.6,720,165 discloses methods whereby the sensitivity of immunoglobulinmolecules to proteolysis is increased by removal of carbohydratemoieties located outside of the hinge region. In another example, USPatent Application Publication 2007/0041979 discloses methods forpreparing antibody molecules with enhanced resistance to proteasesassociated with disease processes by altering the glycosylation state ofthe antibody.

In one aspect of this invention, immunoglobulin from colostrum isfractionated on the basis of displayed carbohydrate to provide apreparation of antibody with improved stability to degradation in thedigestive tract. It is known that bovine colostrum can be fractionatedbased on binding to jacalin. Jacalin is a lectin that recognizesalpha-O-glycoside of the disaccharide Thomsen-Friedenreich antigen (Galbeta1-3GalNAc), even in its sialylated form {Takahashi et al., 2006,Biochem Biophys Res Commun, 350, 580-7}. The jacalin-binding fraction isenriched in IgG1 and has increased stability to pepsin digestioncompared to the non-binding fraction {Porto et al., 2007, J Dairy Sci,90, 955-62}. Fractionation of colostrum based on jacalin binding hasbeen explored in order to develop colostrum preparations that are moreeffective for feeding newborn calves. In one aspect of this invention,colostrum from immunized cows is fractionated for use as a humantherapeutic.

Stabilized antibodies may be generated by the addition of moieties thatsterically block the region or regions of the antibody molecule that aresusceptible to enzymatic digestion. Preferably, these moieties are addedto the hinge region. Stabilized antibodies may be generated by theaddition of moieties that induce conformational changes in the antibodymolecule such that the antibody is not susceptible to enzymatic attack.Stabilized antibodies may be generated by increasing or altering theexpression of carbohydrates such as N-linked or O-linked carbohydratesthat are inherently present on the antibody molecule due to normalcellular processes. Carbohydrates added to the hinge region arepreferably O-linked. Carbohydrates added outside of the hinge region arepreferably N-linked. Stabilized antibodies may be generated by addingN-linked or O-linked carbohydrates to the antibody molecule in vitroafter the antibody has been synthesized. The moieties that conferstability may also be artificial entities such as polyethylene glycol.

Stabilized antibodies may be generated by the addition of one moietythat increases stability to gastric or oral degradation. Stabilizedantibodies may be generated by the addition of multiple moieties thatincrease stability to gastric or oral degradation.

In one embodiment, antibody is fractionated based on the amount or typeof carbohydrate moiety and the stability of the isolated fractions togastric degradation or to degradation in the oral cavity are determined.Antibody may be fractionated using lectin-based chromatography or anyother suitable technique known to those skilled in the art. These dataare used to identify glycosylation patterns associated with stability.

In one embodiment the invention provides a method of enhancing thestability of an antibody to oral or gastric degradation by fractionatingan antibody preparation based on binding to at least one lectin specificfor at least one carbohydrate wherein the binding of that lectin hasbeen shown to correspond with stability to gastric or oral degradation.

In one embodiment of this invention, a preparation of antibody ischaracterized based on the amount or type of glycan moiety for thepurpose of predicting the proportion of antibody in the preparation withincreased stability to gastric degradation or to degradation in the oralcavity. This characterization of antibody may be for the research anddevelopment purposes, for the purpose of setting specifications duringthe development process or for the purpose of in-process testing,release testing, or any other testing required for the manufacture of acommercial product. Characterization of glycan expression may bedetermined using any suitable technique known to those skilled in theart, including, but not limited to, ELISA, Western blotting, NMR,chromatography, electrophoresis, and mass spectrophotometry, includingLC-MS, LC-MS/-MS, MALDI-TOF, TAMNDEM-MS, FTMS.

In one embodiment of this invention, antibody is collected from the milkor colostrum of immunized animals, preferably from the milk or colostrumof immunized cows. In one embodiment of this invention, bovinecolostrum-derived antibody is fractionated based on binding to thelectin jacalin. In one embodiment of this invention, bovinecolostrum-derived antibody is characterized based on the ability of theantibody to bind to the lectin jacalin. In one embodiment the inventionprovides a method of enhancing the stability of an antibody to oral orgastric degradation comprising the steps of:

a) collecting antibodies from the milk or colostrum of cow immunizedwith an antigen;

b) fractionating the antibodies of step (a) based on binding of theantibodies to the lectin jacalin; and optionally

c) assaying the fractions collected in step b), i) for stability togastric or oral degradation, or ii) for the presence of carbohydratesassociated with stability to gastric or oral degradation, or iii) forspecific glycoforms associated with stability to gastric or oraldegradation; and

d) selecting the fractions assayed comprising antibodies that arestabilized to gastric or oral degradation.

In one embodiment, bovine colostral immunoglobulin is fractionated usinglectins specific for particular glycans and the fractions are assayedfor their stability to gastric degradation and for their stability inthe oral cavity. Lectins to be used in this invention include, but arenot limited to, those isolated from Agaricus bisporus, Amaranthuscaudatus, Artocarpus heterophyllus, Artocarpus integrifolia, Griffoniasimplicifolia lectin I, Griffonia simplicifolia lectin II, Griffoniasimplicifolia I B4, Bauhinia purpurea alba, Codium fragile, Daturastramonium, Dolichos biflorus, Erythrina coralldendron, Euonymoseuropaeus, Glycine max, Helix aspersa, Helix pomatia, Hippeastrumhybrid, Lotus tetragonolobus, Lycopersicon esculentum, Maclura pomifera,Narcissus pseudonarcissus, Phaseolus vulgaris L, Phaseolus vulgaris E,Phytolacca Americana, Pisum sativum, Psophocarpus tetragonolobus I,Solanum tuberosum, Sophora japonica terminal, Maackia amurensis, andWisteria floribunda.

In one embodiment, stabilized antibody is generated by treatment withenzymes that add carbohydrate moieties to the antibody molecule.Suitable enzymes include, but are not limited to, O-GlcNAc-transferase,beta-1,4-galactosyltransferase, alpha-2,3-sialyltransferase andbeta-1,4-N-acetylglucosaminyltransferase III. A further list of enzymessuitable for use in this invention is included in US Patent ApplicationPublication 2007/0041979 and is incorporated herein. In one embodiment,the invention provides a method of enhancing the stability of anantibody to oral or gastric degradation comprising the steps of:

a) providing an antibody; and

b) reacting the antibody with enzymes that add carbohydrate moieties tothe antibody molecule wherein the carbohydrate moieties increasestability of the antibody to oral or gastric degradation.

In one embodiment of this invention, stabilized antibodies are generatedby engineering antibodies to express the hinge region sequence frombovine IgG1. Such antibodies are produced using methods described inU.S. Pat. No. 5,677,425. The hinge region of bovine IgG1 links the CH1domain and the CH2 domain. Two allelic variants of bovine IgG1 aresuitable for use in this invention: IgG1a: DPRCKTTCDCCPPPELPGG andIgG1b: DPTCKPSPCDCCPPPELPGG. A third allelic variant, IgG1c:DPRCKRPCDCCPPPELPGG would not be suitable for use in this invention dueto the lack of serine and threonine residues in the hinge region.

In one embodiment, stabilized antibodies are generated by treating hostanimals or cell lines with hormones or other reagents to induce a changein immunoglobulin glycosylation. Although not intended to limit theinvention through a particular mechanism, some of the stability ofcolostral immunoglobulin may result from changes in glycosylationinduced by hormones or other factors associated with pregnancy or withlactation. These hormones or related reagents may be used to treatfemale animals, preferably pregnant animals, before or duringparturition. Such hormones include, but are not limited to, adrenalglucocorticoids, thyroid hormones, prolactin, progesterone, estrogen,insulin and IGF-1, either alone or in combination.

In one embodiment, the invention provides a method of enhancing thestability of an antibody to degradation in the oral cavity or gastricdegradation comprising the steps of:

a) treating an animal with a an agent that induces a change inimmunoglobulin glycosylation; and

b) collecting antibody derived from the animal treated in step (a).

In another embodiment, the invention provides a method of enhancing thestability of an antibody to degradation in the oral cavity or gastricdegradation comprising the steps of:

a) treating an animal with a an agent that induces a change inimmunoglobulin glycosylation;

b) collecting antibody derived from the animal treated in step (a);

c) fractionating the antibody collected in step b; and

d) assaying the fractions collected in step c), i) for stability togastric or oral degradation, or ii) for the presence of carbohydratesassociated with stability to gastric or oral degradation, or iii) forspecific glycoforms associated with stability to gastric oraldegradation.

The hormones or related reagents described above may also be used totreat cell lines used for the expression of immunoglobulin molecules toinduce a change in immunoglobulin glycosylation that results in thegeneration of stabilized antibodies. Such hormones include, but are notlimited to, adrenal glucocorticoids, thyroid hormones, prolactin,progesterone, estrogen, insulin and IGF-1, either alone or incombination. Although not intended to limit this invention to anyparticular cell line, monoclonal antibodies are currently produced usingthe following cell lines: Chinese Hamster Ovary (CHO), Mouse Myeloma(NSO, Sp2/0), Monkey Kidney (COS) and Baby Hamster Kidney (BHK) and allof these cell lines would be suitable in the use of this invention. Insome aspects of this invention, prior to hormone treatment, cell linesmay be transfected with the gene encoding O-GlcNAc transferase, orsimilar enzymes that facilitate appropriate glycosylation, includingO-linked glycosylation of the antibody molecule.

In one embodiment, the invention provides a method of enhancing thestability of an antibody to degradation in the oral cavity or gastricdegradation comprising the steps of:

a) contacting cell lines that are genetically engineered to produceimmunoglobulin with an agent that induces a change in immunoglobulinglycosylation; and

b) collecting antibody produced by the cell line of step (a).

In another embodiment the invention provides a method of enhancing thestability of an antibody to degradation in the oral cavity or gastricdegradation comprising the steps of:

a) contacting cell lines that are genetically engineered to produceimmunoglobulin with an agent that induces a change in immunoglobulinglycosylation;

b) collecting antibody produced by the cell line of step (a);

c) fractionating the antibody collected in step (b);

d) assaying the fractions of step (c) for i) stability to gastric ororal degradation or ii) for the presence of carbohydrates associatedwith stability to gastric or oral degradation, or iii) for specificglycoforms associated with stability to gastric oral degradation; andoptionally;

e) selecting the fractions comprising antibody that is stabilized togastric or oral degradation. In one embodiment, methods may furtherinclude cells that are further genetically engineered to comprise genesencoding transferases that facilitate glycosylation of theimmunoglobulin such as O-linked or N-linked glycosylation.

There is significant heterogeneity in levels and patterns of antibodyglycosylation. This heterogeneity is observed in monoclonal antibodyproduced in tissue culture. This heterogeneity is also observed inpolyclonal antibodies isolated from animals. As described in thisapplication, differences in antibody glycosylation can affect thestability of antibodies to gastric digestion or to degradation in theoral cavity and differences in antibody glycosylation can further affectthe mucosal permeability of antibodies. Therefore, different lots ofantibody, whether isolated from animals or from cultured cells, arelikely to display different levels of stability in the GI tract anddifferent levels of mucosal permeability. In manufacturing therapeuticantibodies for topical application to the GI tract, variability inlevels of stability or permeability would result in clinical materialswith inconsistent potency. As a result, there is a need for analyticalassays to test the glycosylation patterns of antibodies where thoseglycosylation patterns predict the stability of the antibodies in thedigestive tract. There is also a need for methods to be used to applythese assays to the testing of different lots of antibody for thepurpose of accepting or rejecting the particular lot of antibody in themanufacture of a drug product. It is also possible that during themanufacture of an antibody-based drug product (in steps including butnot limited to purification, concentration, buffer exchange,lyophilization, spray drying, formulation and storage) changes in thepattern or level of antibody glycosylation may occur. As a result, thereis a need for analytical assays to test the glycosylation patterns ofantibodies to use in in-process testing, in release testing, and instability assays.

In one embodiment of this invention antibody is tested for enhancedstability to gastric or oral degradation and/or enhanced mucosalpermeability comprising the step of collecting an antibody preparationfrom an antibody source (e.g. milk or serum of immunized animal ormonoclonal antibody cell culture or antibody-based final drug product),and testing for a property associated with enhanced stability to gastricor oral degradation (e.g. the amount and presence of O-linkedglycosylation). In one embodiment, the antibody is tested for a propertyassociated with enhanced stability or mucosal permeability in thedigestive tract and compared to a standardized antibody preparationknown to have enhanced stability or mucosal permeability the digestivetract. In one embodiment of this invention, samples from specific lotsof milk or colostrum from immunized animals are tested to quantify theamount and pattern of glycosylation, where the glycosylation pattern isassociated with the stability of the antibody in the digestive tractand/or with mucosal permeability e.g. the amount and presence ofO-linked glycosylation. The test results are used to determine whetherthose lots should be accepted for inclusion in a product. In oneembodiment of this invention, samples from specific lots of serum fromimmunized animals are tested to quantify the amount and pattern ofglycosylation where the glycosylation pattern is associated with thestability of the antibody in the digestive tract and/or with mucosalpermeability. The test results are used to determine whether those lotsshould be accepted for inclusion in a product. In one embodiment of thisinvention, samples from specific lots of cell supernatant from cellcultures expressing monoclonal antibodies are tested to quantify theamount and pattern of glycosylation where the glycosylation pattern isassociated with the stability of the antibody in the digestive tractand/or with mucosal permeability. The test results are used to determinewhether those lots should be accepted for inclusion in a product. In oneembodiment of this invention, in-process samples collected during themanufacture of an antibody product are tested to quantify the amount andpattern of glycosylation where the glycosylation pattern is associatedwith the stability of the antibody in the digestive tract and/or withmucosal permeability. The test results are used to determine whether themanufacturing process is within the pre-defined specifications andwhether the antibody should be accepted or rejected. In one embodimentof this invention, samples are collected from the final drug substanceor drug product that results from the manufacture of an antibody productare tested to quantify the amount and pattern of glycosylation where theglycosylation pattern is associated with the stability of the antibodyin the digestive tract and/or with mucosal permeability. The testresults are used to determine whether the drug substance or drug productmeets specifications and can be released for the final commercialproduct.

Stabilized antibodies may be used in the oral cavity for the preventionof dental caries and for the treatment or prevention of periodontaldisease as described in U.S. Pat. Nos. 5,759,544; 4,689,221; 4,324,782;4,693,888; 4,725,428; 6,143,330; 5,240,704 and 5,352,446, for thecontrol of microorganisms, including bacteria, protozoa, parasites,viruses and fungi, or for the control of inflammation through the use ofantibodies specific for cytokines or chemokines, or receptors forcytokines or chemokines Stabilized antibodies used in the oral cavitymay be specific for receptors or other antigens expressed on the apicalsurface of the oral cavity, against receptors or other antigensexpressed on the basolateral surface of the mucosal barrier of the oralcavity, or against receptors or other antigens expressed in the mucosa,submucosa, or any other region of the body accessible to topicallyapplied antibody. Such antibodies may be used for the treatment ofinfections of the oral cavity or diseases of the oral cavity, includingbut not limited to mucositis, cancers of the oral cavity, nicotinicstomatitis, leukoplakia, hairy tongue, recurrent aphthous stomatitis,geographic tongue, denture stomatitis, gastroesophageal reflux,eosinophilic esophagitis and lichen planus. Antibodies may also beapplied topically to the oral cavity as a diagnostic reagent asdescribed in U.S. Pat. No. 7,175,430.

Many of the proteases present in the oral cavity are of bacterialorigin. In one embodiment of this invention, a topical antibiotic isadministered to the oral cavity prior to topical administration ofantibody. In one embodiment of this invention, protease inhibitors areadministered to the oral cavity prior to and/or concurrently withtopical administration of antibody. Microbes in the oral cavity alsoproduce hydrolases that remove carbohydrate from antibody, thus makingit more susceptible to proteolytic degradation. In one embodiment ofthis invention, hydrolase inhibitors are administered to the oral cavityprior to and/or concurrently with topical administration of antibody.Antibiotics, protease inhibitors and hydrolase inhibitors may be givenin combination.

Stabilized antibodies may be used in the GI tract for the treatment orprevention of diseases, including but not limited to bacterial, viral orparasitic infections of the gastrointestinal tract, cancers of thegastrointestinal tract, inflammation of the gastrointestinal tract as aresult of injury, surgery, radiation, infection or autoimmune disease.

Stabilized antibodies are useful in the modulation of apical receptorsin the digestive tract, including nutrient receptors, nutrienttransporters, pattern recognition receptors, chemokine receptors,cytokine receptors, bile salt transporters, inorganic ion transporters,mineral transporters, peptidases, saccharases, and growth factorreceptors.

Stabilized antibodies are useful in the treatment or prevention of foodallergies or intolerances, including celiac disease. In one embodiment,a stabilized antibody for treatment of celiac disease is specific forgluten or gluten derived peptides.

Stabilized antibodies are useful in modulating the function ofreceptors, cytokines, chemokines or similar mediators expressed in thelumen of the digestive tract or, in the case of a disease or conditionthat renders the digestive tract permeable to topically appliedantibodies, in modulating the function of receptors, cytokines,chemokines or similar mediators expressed in the portions of the bodybelow the mucosal barrier that are accessible to the antibody.Stabilized antibodies are useful in the treatment of immunodeficiency.

When combined with a suitable delivery vehicle, stabilized antibodiesare useful for systemic dosing.

It is understood by one skilled in the art that antibody fragments arealso subject to degradation in the digestive tract and may be processedusing any of the suitable techniques described in this application toincrease their stability to degradation in the digestive tract. It isunderstood by one skilled in the art that molecules designed to mimicthe activity of antibodies are also subject to degradation in thedigestive tract and may be processed using any of the suitabletechniques described in this application to increase their stability todegradation in the digestive tract.

Antibody Administration to Patients with Compromised Mucosal Barrier inthe Digestive Tract

In the normally functioning digestive tract, intact protein cannot crossthe mucosal barrier. Small amounts of protein are taken up as part ofantigen sampling by the immune system, but this protein is degradedduring antigen processing and does not retain activity. As a result,topical application of an antibody therapeutic to the digestive tractcan only access target antigens that are expressed within the lumen ofthe digestive tract or on the luminal surface of the digestive tract.However, in some disease states, the integrity of the barrier functionis compromised. In this invention, this altered barrier function isexploited to permit therapeutic antibodies applied to the oral cavity orthe lumen of the digestive tract to penetrate the mucosal barrier.

There are 4 elements that bear on the permeability of the digestivetract and the ability of topically applied antibody to penetrate themucosal barrier. First, the area of the digestive tract is an importantfactor, as the mucosal barrier is made up of different cell types atdifferent portions of the digestive tract. The hard palate and gingivain the mouth are lined with keratinized, stratified, squamousepithelium. The soft palate, floor of mouth, cheek, underside of tongueand inside of lips are lined with non-keratinized stratified squamousepithelium. The tongue is a complex mixture of keratinized andnon-keratinized stratified squamous epithelium. The oropharynx is linedwith a non-keratinized, stratified, squamous epithelium. The esophagusis lined with stratified, squamous epithelium and has numerous mucusglands. From the lower esophageal sphincter down to the anus, there is asingle contiguous layer of epithelial cells joined by tight junctions.The small intestine, consisting of the duodenum, the jejunum and theileum, contains numerous finger-like projections called villi. The basesof the villi contain crypts with undifferentiated stem cells. Theepithelium consists mainly of simple columnar epithelial cells,absorptive cells and mucus-secreting goblet cells. The large intestine,consisting of the cecum, colon (ascending, transverse, descending, andsigmoid), rectum, and anal canal, differs from the small intestine inthat it does not contain permanent folds or villi. These various tissuesare known to differ in their permeability to both small molecules and toproteins {Squier, 1991, Crit Rev Oral Biol Med, 2, 13-32}.

Second, the nature of the insult that causes the barrier defect is animportant element. Changes in permeability may result from trauma,irritation, injury from chemicals, radiation or burn, exposure to bile,ischemia, reperfusion injury, inflammation, or infection. Increasedpermeability can result from frank destruction of the mucosa, as in thecase of acute trauma (although it should be noted that epithelialrestitution begins in minutes, and fibrin clots and otherpseudomembranes form rapidly over traumatic ulcers; thus, even antibodyaccessibility to open wounds necessitates antibody transit throughbiological barriers). Increased permeability of the digestive tract canoccur through increased paracellular or transcellular transport, whereparacellular transport is the transport of material through the tightjunctions of the epithelium and transcellular transport is the transportof material through the cells of the epithelium. Both of these processesare highly regulated and affected differently by different factors. Forexample, paracellular transport is differentially affected by theinflammatory cytokines TNF, TGF-beta and IFN-gamma (IFN-γ) and byinfection with enteropathogenic E. coli {Shen and Turner, 2006, Am JPhysiol Gastrointest Liver Physiol, 290, G577-82}. In another example, adirect comparison of traumatic ulcers and recurrent aphthous ulcersrevealed that they healed at different rates and were characterized bydifferent patterns of expression of inflammatory mediators {Natah etal., 2000, J Oral Pathol Med, 29, 19-25}.

Third, the underlying health of the individual and tissue that havesuffered the insult is an important element, because it will affect thehealing process. There are at least 3 mechanisms involved in healing abreach in the mucosal barrier {Sturm and Dignass, 2008, World JGastroenterol, 14, 348-53}. As per the first mechanism, epithelialrestitution begins within minutes, with epithelial cells adjacent to theinjured surface migrating into the wound. Second, epithelial cellproliferate to replenish the decreased cell pool. Third,undifferentiated epithelial cells mature and differentiate to maintainthe many functional activities of the mucosal epithelium. The healing ofthe injury is regulated by cytokines, growth factors, adhesionmolecules, neuropeptides and phospholipids. While inflammation is anessential element in the repair of any wound, inflammation may alsointerfere with these processes, particularly the early stages ofepithelial restitution. Therefore, the abnormal immune system found inindividuals with autoimmune disease, will alter the rate and ability ofthe lesion to heal. Similarly, the presence of chronic inflammation willalter the rate and ability of the lesion to heal.

Fourth, the nature of the antibody particularly the glycosylationpattern of the antibody, will affect the ability of the antibody tocross a damaged mucosal membrane. Glycosylation of the antibody willaffect both its ability to interact with membrane-associated proteins,and its overall charge. The charge on the protein may affect the abilityof the antibody to cross the mucosal membrane. In one aspect of thisinvention, antibodies with enhanced mucosal permeability are preferredfor the treatment of patients with compromised mucosal barriers.

Mucositis

Mucositis, also known as stomatitis, can occur as a result ofchemotherapy or radiation therapy, either alone or in combination. Forthe purposes of this application, mucositis also includes damage causedby exposure to radiation outside of the context of radiation therapy.Chemotherapeutic agents which may induce mucositis when used alone or incombination include, but are not limited to, platinum, cisplatin,carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide,chlorambucil. azathioprine, mercaptopurine, vincristine, vinblastine,vinorelbine, vindesine, etoposide and teniposide, paclitaxel, docetaxel,irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate,teniposide, 5-fluorouracil, leucovorin, methotrexate, gemcitabine,taxane, leucovorin, mitomycin C, tegafur-uracil, idarubicin,fludarabine, mitoxantrone, ifosfamide and doxorubicin. Additional agentsinclude inhibitors of mTOR (mammalian target of rapamycin), includingbut not limited to rapamycin, everolimus, temsirolimus and deforolimus.

As used herein “to treat”, “treating” or “treatment” as it relates tomucositis includes prophylactic and preventative treatment as well astreatment of ongoing disease. For the prevention of mucositis, forexample, the antibodies of the invention can be administered prior toinitiation of a cycle, for example, of chemotherapy and/or radiationtherapy. Alternatively, the antibodies can be administered concurrentlywith, a cycle, for example of chemotherapy and/or radiation therapy.Alternatively, the antibodies can be administered prior to andconcurrently with a cycle, for example, of chemotherapy and/or radiationtherapy. For the treatment of mucositis, for example, the antibodies ofthe invention can be administered concurrently with a cycle, forexample, of chemotherapy and/or radiation therapy. Alternatively, theantibodies can be administered following the completion of a cycle, forexample, of chemotherapy and/or radiation therapy. Alternatively, theantibodies can be administered concurrently with and following thecompletion of a cycle, for example, chemotherapy and/or radiationtherapy. The antibodies in this invention can be used in combinationwith other local or systemic therapies that are in use or will bedeveloped for the treatment or prevention of mucositis.

In one preferred embodiment, anti-TNF antibodies are used to treat orprevent mucositis. In one embodiment, the invention provides a method oftreating mucositis in a patient comprising administering to the patienta composition comprising a therapeutically effective amount of anantibody specific for tumor necrosis factor (TNF). In one embodiment,the anti-TNF antibody is a stabilized antibody of the invention and/oris an antibody with enhanced mucosal permeability. In anotherembodiment, the anti-TNF antibody is administered topically to the oralcavity, or topically to the GI tract as described herein. In oneembodiment, the anti-TNF antibody is administered by parenteraladministration. In one embodiment, the anti-TNF antibody is a polyclonalantibody, a monoclonal antibody, a humanized antibody, a chimerizedantibody, or an antibody fragment or synthetic molecule designed tomimic the function of an antibody as described herein. In one aspect ofthis embodiment, the anti-TNF antibody is a polyclonal antibody derivedfrom the milk or colostrum of a cow.

Other Diseases Associated with Altered Permeability of the DigestiveTract

In one embodiment, the invention provides a method of delivering anantibody to the oral cavity of a patient comprising topicallyadministering the antibody to the oral cavity of the patient, whereinthe mucosal barrier of the patient's oral cavity is compromised suchthat it is permeable to the antibody and wherein the antibody isdirected at targets expressed below the mucosal barrier of the oralcavity. In one embodiment, a topical antibiotic, a protease inhibitor,hydrolase inhibitor or any combination thereof is administered to theoral cavity prior to, or concurrently with the topical administration ofantibody. In one embodiment, the antibody is a stabilized antibody ofthe invention. In one embodiment, the antibody is an antibody withenhanced mucosal permeability. In one embodiment, the antibody isadministered by buccal, gingival or sublingual administration in asuitable dosage form for such administration. Suitable dosage forms foruse in the oral cavity include but are not limited to: buccal patches,buccal tape, mucoadhesive films, sublingual tablets, lozenges, wafers,chewable tablets, quick or fast dissolving tablets, effervescenttablets, a buccal or sublingual solid, granules, sprinkles, pellets,beads, powders, suspensions a mouthwash, gels.

In one embodiment, the invention provides a method of delivering anantibody to the digestive tract of a patient comprising contacting theantibody with the digestive tract of the patient wherein the mucosalbarrier of the patient's digestive tract is compromised such that it ispermeable to the antibody, wherein the antibody is directed at targetsexpressed below the mucosal barrier and wherein the patient is notsuffering from a chronically dysregulated immune system as a result ofimmaturity or an autoimmune disease that targets the digestive tract. Asused herein, a “chronically dysregulated immune system as a result ofimmaturity” includes but is not limited to conditions such asnecrotizing enterocolotis. As used herein the term “autoimmune diseasesthat target the GI tract” includes but is not limited to inflammatorybowel disease (IBD), diabetes, and systemic lupis erythematoses (SLE).

In one embodiment, the invention provides a method of delivering astabilized antibody to the digestive tract of a patient comprisingcontacting the antibody with the digestive tract of the patient whereinthe patient's digestive tract is compromised such that it is permeableto the antibody, wherein the antibody is directed at targets expressedbelow the mucosal surface. In one embodiment, the stabilized antibody isenriched for least one carbohydrate moiety. In another embodiment, theantibody is stabilized by adding one or more carbohydrate moieties tothe antibody after the antibody has been synthesized. In one embodimentthe antibody is stabilized by treatment with enzymes to add carbohydratemolecules to the antibody. In one embodiment, the antibody is astabilized antibody that has been genetically engineered to express thehinge region sequence from bovine IgG1.

In accordance with the methods of the invention, the mucosal barrier ofthe digestive tract may be breached or compromised through mechanicaltrauma, including but not limited to dental and oral wounds, esophagealwounds, or surgically induced trauma due to partial gut resection,jejunostomy, ileostomy, colostomy or other surgical procedures. Themucosal barrier of the digestive tract may also be breached by ischemiaor reperfusion injury.

The mucosal barrier of the digestive tract may be breached orcompromised through gross inflammation and/or ulceration, including butnot limited to periodontal disease, aphthous stomatitis bacterial,viral, fungal or parasitic infections of the digestive tract, pepticulcers, ulcers associated with stress or H. pylori infection, damagecaused by esophageal reflux, inflammatory bowel disease, damage causedby cancer of the digestive tract, food intolerance, including celiacdisease, or ulcers induced by NSAIDs or other ingested or systemicallydelivered drugs.

Patients with irritable bowel syndrome have altered intestinalpermeability despite having little or no detectable histological changesin the intestines (Dunlop S P Am J Gastroenterol. 2006 June;101(6):1288-94). Patients with celiac disease have altered intestinalpermeability and characteristic damage to the villi of the smallintestine that is distinguishable from IBD.

Inflammatory bowel disease is thought to result from a dysregulatedimmune response initiated by microbial-host interactions. The immunesystem responds to non-pathogenic commensal bacteria generating chronicinflammation. Similarly, in necrotizing enterocolitis, a stressedunderdeveloped immune system generates an inappropriate response tonormal intestinal bacteria, inducing a potentially fatal form of colitis{Jilling et al., 2006, J Immunol, 177, 3273-82}.

The breach in or compromise of the mucosal barrier of the digestivetract may be one that has been described clinically but where thebiological basis for the barrier defect is not well understood,including but not limited to the loss of gut barrier function associatedwith external burns, trauma, sepsis or shock, irritable bowel syndrome,diabetes (in particular type I diabetes), atopic dermatitis, patientssuffering from autoimmune disorders, including ankylosing spondylitis,Sjogren's syndrome, congestive heart failure, or multiple sclerosis.Infections with pathogens may also cause specific disruptions of barrierfunction.

In some diseases or disorders to which this invention may be applied,altered barrier permeability may be present prior to the development offrank inflammation and/or ulceration and antibodies may be applied atthe time of altered barrier permeability as well as during the time ofinflammation and ulceration. Diseases and disorders which includeincreased permeability prior to inflammation include but are not limitedto mucositis induced by chemotherapy or radiation therapy, inflammatorybowel disease and celiac disease.

In some diseases or disorders to which this invention may be applied,altered barrier permeability may be present at discrete portions of thedigestive tract while frank inflammation and/or ulceration is present atother portions of the digestive tract {Soderholm et al., 2004, Gut, 53,1817-24}. Diseases and disorders which include physically separatedregions of increased permeability and inflammation or ulceration includebut are not limited to Crohn's disease and ulcerative colitis.Antibodies of this invention may be used to access the regions ofaltered permeability as well as the regions of frank inflammation andulceration.

This invention includes the use of antibodies as therapeutics that aredesigned to address the underlying cause of the barrier defect. Suchantibodies may be directed at biological targets that enhance woundhealing, that alter the function of tight junctions, or at other targetsknown now or in the future that affect permeability. Suitable targetsmay include but are not limited to occludin, claudins, junctionaladhesion molecule, ZO-1, E-cadherin, coxackie adenovirus receptor andserine proteases such as elastase that are involved in the release ofclaudins.

This invention includes the use of antibodies as therapeutics that aredesigned to bind to biological targets unrelated to the underlying causeof the barrier defect. Such antibodies may be used to treat or preventdiseases and disorders relating to the same disease state that causedthe barrier defect. Such antibodies may be used to treat or preventdiseases and disorders unrelated to the disease state that caused thebarrier defect.

This invention includes the use of antibodies directed at biologicaltargets expressed on the basal side of the epithelium, targets expressedin the submucosa, target expressed in the lateral intercellular space,targets expressed in the lamina propria, targets expressed in thecentral nervous system, targets expressed in the systemic circulationand targets expressed in any region of the body that is accessible todelivered antibody as a result of damaged barrier function.

This invention includes the use of antibodies that are also known to beeffective when administered by injection or infusion for systemicexposure. For example, systemically administered anti-TNF antibodies areeffective in the treatment of inflammatory bowel disease {Kozuch andHanauer, 2008, World J Gastroenterol, 14, 354-77}, recurrent aphthousstomatitis {Vujevich and Zirwas, 2005, Cutis, 76, 129-32}, and Behçet'sdisease {Almoznino and Ben-Chetrit, 2007, Clin Exp Rheumatol, 25,S99-102}. The antibodies of this invention may be more efficacious whenadministered topically or topical application may result in reduced sideeffects. Topical application may also be preferred, even in the absenceof superior efficacy or side effect profile, because of increased easeof administration. This invention includes the use of antibodies thathave not been shown to be effective or are not effective whenadministered by injection or infusion for systemic exposure.

For the treatment of aphthous stomatitis (RAS), the antibodies of theinvention can be administered at the earliest manifestation of an ulcer.Alternatively, the antibodies can be administered on a regular basisthroughout the course of manifestation of the ulcer. Alternatively, theantibodies can be administered on a regular basis to prevent therecurrence of ulcer formation. In one preferred embodiment, theinvention provides a method of treating recurrent aphthous stomatitis(RAS) in a patient comprising administering to the patient by topicalapplication to the oral cavity a therapeutically effective amount of acomposition comprising an antibody specific for TNF (an anti-TNFantibody). In one embodiment the anti-TNF antibody is a stabilizedantibody of the invention. In one embodiment the anti-TNF antibody is anantibody with enhanced mucosal permeability. In another embodimentanti-TNF antibody is administered topically to the oral cavity asdescribed herein. In one embodiment, the anti-TNF antibody is apolyclonal antibody, a monoclonal antibody, a humanized antibody, achimerized antibody, or an antibody fragment or synthetic moleculedesigned to mimic the function of an antibody as described herein.

In one embodiment of the invention, antibodies are not targeted toexogenous agents, where “exogenous agents” are defined as those agentsthat are not synthesized in the body of the animal being treated withthe antibodies. Agents that are synthesized by microorganisms residentin the body of the animal being treated with the antibodies areconsidered exogenous agents. Biological targets of the antibodies of theinvention are preferably endogenous to the organism being administeredthe antibody.

In one embodiment of the invention, antibodies are not targeted toinfectious agents, including viruses, bacteria, fungi, protozoa andparasites.

In one embodiment of the invention, antibodies are not targeted toantigens expressed in the lumen of the digestive tract or on the luminalaspect of the digestive tract epithelium.

In one embodiment of the invention, antibodies specific for inflammatorycytokines, including but not limited to TNF, TNF-kappa, IL-6, Ifn-gamma,IL-1 beta, IL-12, IL-13, IL-23, IL-17 and IL-2 are applied topically tothe digestive tract of a patient with increased permeability of thedigestive tract to prevent the development of frank ulceration orinflammation due to chemotherapy or radiation therapy.

In one embodiment of the invention, antibodies specific for inflammatorycytokines, including but not limited to TNF, TNF-kappa, IL-6, Ifn-gamma,IL-1 beta, IL-12, IL-13, IL-23, IL-17 and IL-2 are applied topically tothe digestive tract of a patient with increased permeability of thedigestive track to prevent the development of frank ulceration orinflammation due to autoimmune disease, including inflammatory boweldisease.

In one embodiment of the invention, antibodies specific for Toll-likereceptors that are expressed on the basolateral face of mucosalepithelial cells are applied as a therapeutic agent to the digestivetract of a patient with an intestinal inflammatory disease.

In one embodiment of the invention, antibodies specific for inflammatorycytokines, including but not limited to TNF, TNF-kappa, IL-6, Ifn-gamma,IL-1 beta, IL-12, IL-13, IL-23, IL-17 and IL-2 are applied as atherapeutic agent to the digestive tract of a patient with irritablebowel syndrome.

In one embodiment of the invention, antibodies directed at entericneurotransmitters or their receptors or transporters expressed below themucosal barrier of the digestive tract, including receptors forserotonin that are expressed in the gut (5-HT1A, 5-HT1B/B, 5-HT2A,5-HT2B, 5-HT3, 5-HT4, 5-HT7, 5-HT1P {De Ponti, 2004, Gut, 53, 1520-35})are used as pharmaceutical agents in patients with increased digestivetract permeability.

In one embodiment of the invention, antibodies directed at peptides thatregulate food intake or the receptors for such peptides are used aspharmaceutical agents in patients with increased digestive tractpermeability. Such peptides include but are not limited to CCK, GLP1,GIP, oxyntomodulin, PYY3-36, enterostatin, APOAIV, PP, amylin, GRP andNMB, gastric leptin and ghrelin {Cummings and Overduin, 2007, J ClinInvest, 117, 13-23}.

In one embodiment of the invention, antibodies directed at epidermalgrowth factor receptor on colorectal cancer cells are used astherapeutic agents in patients with increased digestive tractpermeability.

In one embodiment of the invention, patients suffering from radiationexposure, trauma, burn, shock or sepsis are treated orally withantibodies directed against infectious agents, inflammatory cytokines,including but not limited to TNF-alpha. In one embodiment, the antibodyis not targeted to TNF when the disease is Crohn's Disease or UlcerativeColitis.

Patients suitable for application of this invention are identified bydirect measurement of digestive tract permeability, by diagnosis with adisease associated with increased digestive tract permeability, bydetection of a genetic marker or biomarker associated with increaseddigestive tract permeability, by known or presumed exposure to an agentknown to induce increased digestive tract permeability or by geneticrelationship with an individual known to have or be at risk forincreased digestive tract permeability. U.S. Pat. No. 6,037,330 teachesmethods to detect damage to specific portions of the digestive tractthat manifest as increased permeability.

It is known in the art that the permeability of the digestive tract canbe intentionally increased for the purposes of drug delivery. Permeationenhancers are available, including chitosan, poly-L-arginine andCarbopol, which have been used to enhance buccal absorption ofpharmaceuticals. U.S. Pat. No. 5,849,322 teaches methods to enhancebuccal delivery of therapeutics. U.S. Pat. No. 5,665,389 teaches theintentional enhancement of intestinal permeability for the purposes ofdelivering insulin to the systemic circulation. In none of theseteachings has the increased permeability associated with the diseasestate been exploited for the purposes of delivering an antibody, aprotein, or any other therapeutic agent. The methods of the presentinvention include the use of such permeation enhancers to enhance thedelivery of antibody to the digestive tract.

As discussed earlier, the nature of the antibody particularly theglycosylation pattern of the antibody, will affect the ability of theantibody to cross a damaged mucosal membrane. Glycosylation of theantibody will affect both its ability to interact withmembrane-associated proteins, and its overall charge. The charge on theprotein may affect the ability of the antibody to cross the mucosalmembrane. In one aspect of this invention, antibodies with definedglycosylation patterns conferring enhanced mucosal permeability on theantibodies are preferred for the treatment of patients with compromisedmucosal barriers.

In one embodiment, the invention provides antibodies with enhancedmucosal permeability. Preferably the antibody comprises enhanced mucosalpermeability in a compromised digestive tract. Antibodies comprisingenhanced mucosal permeability include monoclonal antibodies, polyclonalantibodies, and/or any other antibody as described herein. In oneembodiment, the antibody is enriched for at least one glycoform thatconfers enhanced mucosal permeability. In one embodiment, the glycoformcontains oligomannose or sialic acid. In one embodiment, the glycoformdoes not contain oligomannose or sialic acid. In one embodiment, theantibody has been treated with enzymes to add or remove carbohydratesthat confer enhanced permeability after the antibody has beensynthesized. In another embodiment, the antibody having enhanced mucosalpermeability is also a stabilized antibody comprising enhanced stabilityto oral or gastric degradation as described herein.

In one embodiment, the invention provides a method of delivering anantibody to the digestive tract of a patient comprising contacting theantibody with the digestive tract of the patient wherein the mucosalbarrier of the patient's digestive tract is compromised such that it ispermeable to the antibody, and wherein the antibody is directed attargets expressed below the mucosal barrier and wherein the antibodycomprises a glycosylation pattern that enhances the antibody's abilityto cross the permeable mucosal barrier of the compromised digestivetract. In one embodiment, antibody is tested for enhanced permeability,particularly enhanced permeability in a compromised digestive tract,comprising the step of collecting an antibody preparation from anantibody source (e.g. milk or serum of immunized animal or monoclonalantibody cell culture) and testing the antibody preparation for aproperty associated with enhanced permeability in a compromiseddigestive tract e.g. the amount and pattern of O-linked glycosylation.In one embodiment of this invention, samples from specific lots of milkor colostrum from immunized animals are tested to quantify the amountand pattern of glycosylation, where the glycosylation pattern isassociated with enhanced mucosal permeability in a compromised digestivetract e.g. the amount and pattern of O-linked glycosylation. In oneembodiment, the antibody is tested for a property associated withenhanced mucosal permeability in a compromised digestive tract andcompared to a standardized antibody preparation known to have enhancedmucosal permeability in a compromised digestive tract.

In one embodiment the invention provides a method of identifyingantibodies having enhanced mucosal permeability in a compromiseddigestive tract comprising the steps of:

a) fractionating an antibody preparation to provide separate antibodypreparations each with a different pattern of glycosylation; and

b) assaying the antibody preparations of step (a) for enhanced abilityto cross the permeable mucosal barrier of a compromised digestive tract.

In one preferred embodiment, the invention provides a method of treatinginflammatory bowel disease (IBD) or Crohn's disease in a patientcomprising administering to the patient a composition comprising atherapeutically effective amount of an antibody specific for tumornecrosis factor (TNF) wherein the antibody comprises a glycosylationpattern that enhances the ability of the antibody to cross the permeablemucosal barrier of a patient suffering from IBD or Crohn's disease. Inone aspect of this embodiment the antibody is a polyclonal antibodyderived from the milk or colostrum of a cow.

For disorders of the oral cavity, the antibodies of the invention can bedelivered in a mouthwash, rinse, paste, gel, or other suitableformulation. Antibodies of the invention can be delivered usingformulations designed to increase the contact between the activeantibody and the mucosal surface, such as buccal patches, buccal tape,mucoadhesive films, sublingual tablets, lozenges, wafers, chewabletablets, quick or fast dissolving tablets, effervescent tablets, or abuccal or sublingual solid. For disorders of the GI tract, antibody canbe delivered by oral ingestion in the form of a capsule, tablet, liquidformulation or similar form designed to introduce drug to the GI tract.Alternatively, antibody may be administered by suppository or enema fordelivery to the lower GI tract. Such formulations are well known tothose skilled in the art. These routes of administration and dosageforms are discussed in detail herein.

Antibodies

The terms “antibody” or “antibodies” as used herein refer to apolypeptide comprising a framework region from an immunoglobulin gene orfragments thereof that specifically binds and recognizes an antigen. Therecognized immunoglobulin genes include the kappa, lambda, alpha, gamma,delta, epsilon, and mu constant region genes, as well as the myriadimmunoglobulin variable region genes. Light chains are classified aseither kappa or lambda. Heavy chains are classified as gamma, mu, alpha,delta, or epsilon, which in turn define the immunoglobulin classes, IgG,IgM, IgA, IgD and IgE, respectively. Typically, the antigen-bindingregion of an antibody will be most critical in specificity and affinityof binding to a target receptor. An exemplary immunoglobulin (antibody)structural unit comprises a tetramer. Each tetramer is composed of twoidentical pairs of polypeptide chains, each pair having one “light”(about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus ofeach chain defines a variable region of about 100 to 110 or more aminoacids primarily responsible for antigen recognition. The terms variablelight chain (V_(L)) and variable heavy chain (V_(H)) refer to theselight and heavy chains respectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number ofwell-characterized fragments produced by degradation with variouspeptidases that are able to compete with the intact antibody forspecific binding, unless otherwise specified herein. Thus, for example,pepsin digests an antibody below the disulfide linkages in the hingeregion to produce F(ab)′₂, a dimer of Fab which itself is a light chainjoined to V_(H-CH)1 by a disulfide bond. The F(ab)′₂ may be reducedunder mild conditions to break the disulfide linkage in the hingeregion, thereby converting the F(ab)′₂ dimer into an Fab′ monomer. TheFab′ monomer is essentially Fab with part of the hinge region (seeFundamental Immunology (Paul ed., 3d ed. 1993). While various antibodyfragments are defined in terms of the degradation of an intact antibody,one of skill will appreciate that such fragments may be synthesized denovo either chemically or by using recombinant DNA methodology. Thus,the term “antibody”, as used herein, also includes antibody fragmentseither produced by the modification of whole antibodies, or thosesynthesized de novo using chemical or recombinant DNA methodologies(e.g., single chain Fv, complementarity determining region (CDR)fragments, or polypeptides that contain at least a portion of animmunoglobulin that is sufficient to confer specific receptor binding tothe polypeptide) or those identified using phage display libraries (see,e.g., McCafferty et al., Nature 348: 552-554 (1990)).

The terms “monoclonal antibody” or “monoclonal antibodies” as usedherein refer to a preparation of antibodies of single molecularcomposition. A monoclonal antibody composition displays a single bindingspecificity and affinity for a particular epitope of a target receptor.

An “epitope” is the portion of a molecule that is bound by an antibody.An epitope can comprise non-contiguous portions of the molecule (e.g.,in a polypeptide, amino acid residues that are not contiguous in thepolypeptide's primary sequence but that, in the context of thepolypeptide's tertiary and quaternary structure, are near enough to eachother to be bound by an antibody).

The term “polyclonal antibody” as used herein refers to a composition ofdifferent antibody molecules which is capable of binding to or reactingwith several different specific antigenic determinants on the same or ondifferent antigens. The variability in antigen specificity of apolyclonal antibody is located in the variable regions of the individualantibodies constituting the polyclonal antibody, in particular in thecomplementarity determining regions (CDR)1, CDR2 and CDR3 regions.Preferably, the polyclonal antibody is prepared by immunization of ananimal with the target antigen or portions thereof as specified below.Alternatively, the polyclonal antibody may be prepared by mixingmultiple monoclonal antibodies (e.g. Nowakowski, A. et al. 2002. ProcNatl Acad Sci USA 99, 11346-11350 and U.S. Pat. No. 5,126,130) havingdesired specificity to a target receptor.

Polyclonal antibody preparations isolated from the blood, milk,colostrum or eggs of immunized animals typically include antibodies thatare not specific for the immunogen in addition to antibodies specificfor the target receptor. Antibodies specific for the target receptor maybe purified from the polyclonal antibody preparation or the polyclonalantibody preparation may be used without further purification. Thus, theterm “polyclonal antibody” as used herein refers both to antibodypreparations in which the antibody specific for the target receptor hasbeen enriched and to preparations that are not purified. Numeroustechniques are known to those in the art for enriching polyclonalantibodies for antibodies to specific targets. A technology forrecombinant production of highly specific polyclonal antibodies suitablefor prophylactic and therapeutic administration has been developed (WO2004/061104). The recombinant polyclonal antibody (rpAb) can be purifiedfrom a production bioreactor as a single preparation without separatehandling, manufacturing, purification, or characterization of theindividual members constituting the recombinant polyclonal protein.

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity. See, e.g., U.S. Pat. No. 4,816,567 andMorrison, 1985, Science 229:1202-07.

The invention further contemplates the use of molecules intended tomimic antibodies, such as aptamers. The invention also contemplates theuse of “fusion proteins” in which a portion of an antibody molecule isfused to the ligand for the target receptor and thereby made specificfor the target receptor. In another aspect, the present inventionprovides a derivative of an antibody specific for a target antigen. Thederivatized antibody can comprise any molecule or substance that impartsa desired property to the antibody, such as increased half-life in aparticular use. The derivatized antibody can comprise, for example, adetectable (or labeling) moiety (e.g., a radioactive, colorimetric,antigenic or enzymatic molecule, a detectable bead (such as a magneticor electrodense (e.g., gold bead), or a molecule that binds to anothermolecule (e.g., biotin or streptavidin), a therapeutic or diagnosticmoiety (e.g., a radioactive, cytotoxic, or pharmaceutically activemoiety), or a molecule that increases the suitability of the antibodyfor a particular use (e.g., administration to a subject, such as a humansubject, or other in vivo or in vitro uses). Examples of molecules thatcan be used to derivatize an antibody include albumin (e.g., human serumalbumin) and polyethylene glycol (PEG). Albumin-linked and PEGylatedderivatives of antibodies can be prepared using techniques well known inthe art. In one embodiment, the antibody is conjugated or otherwiselinked to transthyretin (TTR) or a TTR variant. The TTR or TTR variantcan be chemically modified with, for example, a chemical selected fromthe group consisting of dextran, poly(n-vinyl pyrrolidone), polyethyleneglycols, propropylene glycol homopolymers, polypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols and polyvinyl alcohols.

Derivatized antibodies are also suitable for in-vivo or in-vitrodetection of expression of a target. In one preferred embodiment, anantibody derivatized with a physiologically acceptable label detectableby standard imaging equipment such as ultrasound, is used for in-vivodiagnostic imaging to detect aberrant expression of a target. Suchdiagnostic techniques are useful in identifying patients who haveelevated expression, activation or activity of a target associated withone or more diseases thereby identifying patients who may benefit mostfrom treatment with an antibody of the invention.

The present invention further comprises nucleic acid molecules encodingall or a part of an antibody of the invention, for example, one or bothchains of the antibody of the invention or a fragment, derivative, orvariation thereof. The nucleic acids can be single-stranded or doublestranded and can comprise RNA and/or DNA nucleotides or variants thereof such as peptide nucleic acids. The present invention furthercomprises host cells into which a recombinant expression vector ortransfectoma is introduced and is capable of expressing an antibody ofthe invention or fragment thereof. A host cell can be any prokaryoticcell or eukaryotic cell. Vector DNA can be introduced into a host cellvia conventional transformation or transfection techniques.

In one embodiment, the antibody of the invention is capable of at leastpartially blocking at least one biological activity of a target antigen.In another embodiment, the antibody of the invention has a bindingaffinity (K_(a)) for the target receptor of at least 10⁶. In otherembodiments, the antibody exhibits a K_(a) of at least 10⁷, at least10⁸, at least 10⁹, or at least 10¹⁰. In another embodiment, the presentinvention provides an antibody that has a low dissociation rate from atarget antigen. In one embodiment, the antibody has a K_(off) of 1×10⁻⁴s⁻¹ or lower. In another embodiment, the K_(off) is 5×10⁻⁵ s⁻¹ or lower.It is understood by those skilled in the art that these affinities anddissociation rates refer to average affinities and dissociation rateswhen used to describe polyclonal antibodies. It is further understood bythose skilled in the art that affinity is defined broadly and includesavidity as well as affinity. In another aspect, the present inventionprovides an antibody that inhibits at least one biological activity of atarget antigen. In one embodiment, the antibody has an IC₅₀ of 1000 nMor lower. In another embodiment, the IC₅₀ is 100 nM or lower; in anotherembodiment, the IC₅₀ is 10 nM or lower.

In one embodiment, monoclonal antibodies are preferred. In anotherembodiment polyclonal antibodies are preferred. Monoclonal antibodiesare more controllable, but their specificity is limited. Polyclonalantibodies are more difficult to characterize, but their broadspecificity means that they can interfere with target receptors inseveral different ways. In addition, the manufacture of polyclonalantibodies can be very inexpensive.

Methods of producing polyclonal and monoclonal antibodies that reactspecifically with the target antigens of the invention are known tothose of skill in the art (see, e.g., Coligan, Current Protocols inImmunology (1991); Harlow & Lane, Antibodies, A Laboratory Manual(1988); Goding, Monoclonal Antibodies: Principles and Practice (2d ed.1986); and Kohler & Milstein, Nature 256: 495-497 (1975). Suchtechniques include antibody preparation by selection of antibodies fromlibraries of recombinant antibodies in phage or similar vectors, as wellas preparation of polyclonal and monoclonal antibodies by immunizingsuitable animals (see, e.g., Huse et al., Science 246: 1275-1281 (1989);Ward et al., Nature 341: 544-546 (1989)).

A number of immunogens comprising target antigens or portions of targetantigens may be used to produce antibodies specifically reactive withthe target antigen. For example, an antigenic fragment or proteinportion of a target antigen can be isolated using known procedures.Recombinant protein can be expressed in eukaryotic or prokaryotic cellsas described above, and purified as generally described above.Alternatively, a synthetic peptide derived from a target antigen can beused as an immunogen. Preferably, the peptide is derived from a portionof the target antigen that is expressed extracellularly. The syntheticpeptide may be conjugated to a carrier protein prior to immunization.Naturally occurring protein may also be used either in pure or impureform. The product is then injected into an animal capable of producingantibodies. Animals may also be immunized with cells that have beentransfected with the target antigen or may be immunized with DNAencoding the target antigen. Either monoclonal or polyclonal antibodiesmay be generated accordingly.

Monoclonal antibodies may be obtained by various techniques familiar tothose skilled in the art. Briefly, spleen cells from an animal immunizedwith a desired antigen are immortalized, commonly by fusion with amyeloma cell (see, Kohler & Milstein, Eur. J. Immunol. 6: 511-519(1976)). Alternative methods of immortalization include transformationwith Epstein Barr Virus, oncogenes, or retroviruses, or other methodswell known in the art. Colonies arising from single immortalized cellsare screened for production of antibodies of the desired specificity andaffinity for the antigen, and yield of the monoclonal antibodiesproduced by such cells may be enhanced by various techniques, includinginjection into the peritoneal cavity of a vertebrate host.Alternatively, one may isolate DNA sequences which encode a monoclonalantibody or a binding fragment thereof by screening a DNA library fromhuman B cells according to the general protocol outlined by Huse, etal., Science 246: 1275-1281 (1989).

Methods of production of polyclonal antibodies are known to those ofskill in the art. An appropriate animal is immunized with the proteinusing a standard adjuvant, such as Freund's adjuvant, and a standardimmunization protocol. The animal's immune response to the immunogenpreparation may be monitored by taking test bleeds and determining thetiter of reactivity to target receptor. When appropriately high titersof antibody to the immunogen are obtained, blood is collected from theanimal and antisera are prepared. Further fractionation of the antiserato enrich for antibodies reactive to the protein can be done if desired(see, Harlow & Lane, supra).

Alternatively, eggs can be collected from immunized birds and antibodyis isolated from the yolks of the eggs. Alternatively, milk or colostrumcan be collected from immunized female animals and antibody is isolatedfrom the milk or colostrum.

In one embodiment the antibody is isolated from the yolk of eggs from abird such as a chicken, duck, or goose that has been immunized with atarget antigen and/or peptide or antigenic portion derived from a targetantigen and a suitable adjuvant. In another embodiment, the antibody isisolated from the serum of an animal such as a cow, horse, rabbit, orgoat that has been immunized with an antigen and/or peptide derived froman antigen and a suitable adjuvant.

In one embodiment, the antibody is a polyclonal antibody derived frommilk or colostrum. In one embodiment, the polyclonal antibody is derivedfrom the milk or colostrum of a ruminant such as a cow, goat, sheep,camel or water buffalo. In another embodiment, the antibody is isolatedfrom the milk or colostrum of a human. In a preferred embodiment, thepolyclonal antibody is isolated from the milk or colostrum of a bovine,preferably an immunized cow. Bovine colostrum (early milk) is apreferred source of antibodies for this invention. In cows, antibodydoes not cross the placenta, and thus all passive immunity istransferred to the newborn calf through the milk. As a result, cowssecrete a large bolus of antibody into the colostrum immediately afterparturition and approximately 50% of the protein in colostrum isimmunoglobulin. In the first 4 hours after birth, immunoglobulinconcentrations of 50 mg/ml are typically found in the colostrum {Butlerand Kehrli, 2005, Mucosal Immunology, 1763-1793}, dropping to 25-30mg/ml 24 hours later {Ontsouka et al., 2003, J Dairy Sci, 86, 2005-11}.Colostrum and milk are a uniquely safe source of polyclonal antibody fororal delivery. There is already extensive human exposure to bovineimmunoglobulin, as regular milk contains 1.5 g/L IgG.

In one aspect, the invention provides methods of treating a patientusing the therapeutic compositions of the invention. The term “patient”as used herein refers to an animal. Preferably the animal is a mammal.More preferably the mammal is a human. A “patient” also refers to, forexample, dogs, cats, horses, cows, pigs, guinea pigs, fish, birds andthe like. Thus, the compositions and methods of the invention areequally suitable for veterinary treatments. In one embodiment of theinvention, antibodies are used to treat diseases or disorders ofcompanion animals, work animals or animals raised for food. In oneembodiment of the invention, stabilized antibodies are used to providepassive immunity to newborn animals, preferably to cows, horses, sheepor swine.

The terms “treatment” “treat” and “treating” encompasses alleviation,cure or prevention of at least one symptom or other aspect of adisorder, disease, illness or other condition (collectively referred toherein as a “condition”), or reduction of severity of the condition, andthe like. A composition of the invention need not effect a completecure, or eradicate every symptom or manifestation of a disease, toconstitute a viable therapeutic agent. As is recognized in the pertinentfield, drugs employed as therapeutic agents may reduce the severity of agiven disease state, but need not abolish every manifestation of thedisease to be regarded as useful therapeutic agents. Similarly, aprophylactically administered treatment need not be completely effectivein preventing the onset of a condition in order to constitute a viableprophylactic agent. Simply reducing the impact of a disease (forexample, by reducing the number or severity of its symptoms, or byincreasing the effectiveness of another treatment, or by producinganother beneficial effect), or reducing the likelihood that the diseasewill occur or worsen in a subject, is sufficient. In one embodiment, anindication that a therapeutically effective amount of a composition hasbeen administered to the patient is a sustained improvement overbaseline of an indicator that reflects the severity of the particulardisorder.

The pharmaceutical compositions of the present invention comprise atherapeutically effective amount of an antibody of the present inventionformulated together with one or more pharmaceutically acceptablecarriers or excipients. By a “therapeutically effective amount” of anantibody of the invention is meant an amount of the composition whichconfers a therapeutic effect on the treated subject, at a reasonablebenefit/risk ratio applicable to any medical treatment. The therapeuticeffect is sufficient to “treat” the patient as that term is used herein.

As used herein, the term “pharmaceutically acceptable carrier orexcipient” means a non-toxic, inert solid, semi-solid or liquid filler,diluent, encapsulating material or formulation auxiliary of any type.Some examples of materials which can serve as pharmaceuticallyacceptable carriers are sugars such as lactose, glucose and sucrose;starches such as corn starch and potato starch; cellulose and itsderivatives such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; powdered tragacanth; malt; gelatin; talc; excipientssuch as cocoa butter and suppository waxes; oils such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; glycols such as propylene glycol; esters such as ethyloleate and ethyl laurate; agar; buffering agents such as magnesiumhydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffersolutions, as well as other non-toxic compatible lubricants such assodium lauryl sulfate and magnesium stearate, as well as coloringagents, releasing agents, coating agents, sweetening, flavoring andperfuming agents, preservatives and antioxidants can also be present inthe composition, according to the judgment of the formulator.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the active compounds, the liquid dosage formsmay contain inert diluents commonly used in the art such as, forexample, water or other solvents, solubilizing agents and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethylformamide, oils (in particular, cottonseed, groundnut,corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Compositions for rectal administration are preferably suppositorieswhich can be prepared by mixing the compounds of this invention withsuitable non-irritating excipients or carriers such as cocoa butter,polyethylene glycol or a suppository wax which are solid at ambienttemperature but liquid at body temperature and therefore melt in therectum or vaginal cavity and release the active compound. In oneembodiment, compositions for rectal administration are in the form of anenema.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or: a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents. Solid compositions of asimilar type may also be employed as fillers in soft and hard-filledgelatin capsules using such excipients as lactose or milk sugar as wellas high molecular weight polyethylene glycols and the like.

Although stabilized antibodies have enhanced stability to gastricdegradation, it may be desirable under some conditions to provideadditional levels of protection against gastric degradation. If this isdesired, there are many options for enteric coating (see for exampleU.S. Pat. Nos. 4,330,338 and 4,518,433). In one embodiment, entericcoatings take advantage of the post-gastric change in pH to dissolve afilm coating and release the active ingredient. Coatings andformulations have been developed to deliver protein therapeutics to thesmall intestine and these approaches could be adapted for the deliveryof an antibody of the invention. For example, an enteric-coated form ofinsulin has been developed for oral delivery {Toorisaka et al., 2005, JControl Release, 107, 91-6}.

In addition, the solid dosage forms of tablets, dragees, capsules,pills, and granules can be prepared with other coatings and shells wellknown in the pharmaceutical formulating art. They may optionally containopacifying agents and can also be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain part of theintestinal tract, optionally, in a delayed manner. Examples of embeddingcompositions that can be used include polymeric substances and waxes.

Effective doses will vary depending on route of administration, as wellas the possibility of co-usage with other agents. It will be understood,however, that the total daily usage of the compounds and compositions ofthe present invention will be decided by the attending physician withinthe scope of sound medical judgment. The specific therapeuticallyeffective dose level for any particular patient will depend upon avariety of factors including the disorder being treated and the severityof the disorder; the activity of the specific compound employed; thespecific composition employed; the age, body weight, general health, sexand diet of the patient; the time of administration, route ofadministration, and rate of excretion of the specific compound employed;the timing of delivery of the compound relative to food intake; theduration of the treatment; drugs used in combination orcontemporaneously with the specific compound employed; and like factorswell known in the medical arts.

Particular embodiments of the present invention involve administering apharmaceutical composition comprising an antibody of the invention at adosage of from about 1 mg per day to about 1 g/day, more preferably fromabout 10 mg/day to about 500 mg/day, and most preferably from about 20mg/day to about 100 mg/day, to a subject. In one embodiment, apolyclonal antibody preparation is administered at a dosage of antibodyfrom about 100 mg to about 50 g/day, more preferably from about 500mg/day to about 10 g/day, and most preferably from about 1 g/day toabout 5 g/day, to a subject, wherein the polyclonal antibody preparationhas not been enriched for antibodies specific for the target antigen.

Treatment regimens include administering an antibody composition of theinvention one time per day, two times per day, or three or more timesper day, to treat a medical disorder disclosed herein. In oneembodiment, an antibody composition of the invention is administeredfour times per day, 6 times per day or 8 times per day to treat amedical disorder disclosed herein. In one embodiment, an antibodycomposition of the invention is administered one time per week, twotimes per week, or three or more times per week, to treat a medicaldisorder disclosed herein.

The methods and compositions of the invention include the use of anantibody of the invention in combination with one or more additionaltherapeutic agents useful in treating the condition with which thepatient is afflicted. Examples of such agents include both proteinaceousand non-proteinaceous drugs. When multiple therapeutics areco-administered, dosages may be adjusted accordingly, as is recognizedin the pertinent art. “Co-administration” and combination therapy arenot limited to simultaneous administration, but also include treatmentregimens in which an antibody of the invention is administered at leastonce during a course of treatment that involves administering at leastone other therapeutic agent to the patient.

The following examples are provided for the purpose of illustratingspecific embodiments or features of the invention and are not intendedto limit its scope.

EXAMPLES Example 1 Treatment of Radiation-Induced Mucositis by TopicalApplication of Anti-TNF Antibody

Mucositis was induced in Golden Syrian hamsters (male, 5-6 weeks old, 8animals per group, Charles River Laboratories) using a standardizedacute radiation protocol. A single dose of radiation (40 Gy/dose) wasadministered to all animals on day 0. Radiation was generated with a 160kilovolt potential (15-ma) source at a focal distance of 50 cm, hardenedwith a 0.35 mm Cu filtration system. Irradiation targeted the leftbuccal pouch mucosa at a rate of 2.0 Gy/minute. Prior to irradiation,animals were anesthetized with an i.p. injection of Ketamine (160 mg/ml)and Xylazine (8 mg/ml). The left buccal pouch was everted, fixed andisolated using a lead shield.

Hamsters were administered purified rabbit anti-mouse TNF antibody(BioVision, Mountain View, Calif.) twice a day in the left buccal cheekpouch for 14 days starting on day −1 (day −1 to day 12). Antibody wasadministered in 0.2 ml, the approximate capacity of the cheek pouch. Twodoses of antibody were compared (4.0 μg and 0.4 μg) and a vehicle salinecontrol.

Mucositis was evaluated starting on day 6, and continuing on alternatedays until day 28. Animals were anesthetized and the left cheek poucheverted and photographed. At the end of the study, the images wererandomized and scored in an independent manner by 2 scorers who wereblinded as to the identifiers for each image. The scale ranges from 0for normal, to 5 for severe ulceration as is shown in the accompanyingtable. A score of 1-2 is considered to represent a mild stage of thedisease, while a score of 3-5 is considered to represent moderate tosevere mucositis. On day 28, all animals were sacrificed.

Score Description 0 Pouch completely healthy. No erythema orvasodilation 1 Light to severe erythema and vasodilation. No erosion ofmucosa 2 Severe erythema and vasodilation. Erosion of superficialaspects of mucosa leaving denuded areas. Decreased stippling of mucosa.3 Formation of off-white ulcers in one or more places. Ulcers may have ayellow/gray color due to pseudomembrane. Cumulative size of ulcersshould equal about ¼ of the pouch. Severe erythema and vasodilation 4Cumulative size of ulcers should equal about ½ of the pouch. Loss ofpliability. Severe erythema and vasodilation 5 Virtually all of pouch isulcerated. Loss of pliability (pouch can only partially be extractedfrom mouth

The severity and duration of ulcerative mucositis was less severe inanimals receiving the 4.0 μg dose of anti-TNF antibody than in theanimals treated with saline vehicle control. Vehicle-treated animalsexhibited a peak mucositis score of 3.6 on day 16 which had decreased toan average score of 2.1 by day 28. The group treated with the 4.0 μgdose of antibody had an average peak score of 3.3 on day 18 thatdecreased to 1.5 by day 28. The 0.4 μg/dose anti-TNF treatment failed todemonstrate any efficacy in the treatment of oral mucositis compared tothe saline control.

The significance of the differences between the vehicle control and thetreated groups was assessed by comparing the number of days with anulcer (i.e. a score of 3 or higher) using a chi-squared (χ2) test. Thevehicle control group had scores of 3 or higher on 58.3% of the animaldays evaluated. The group treated with the low 0.4 μg/dose of anti-TNFhad scores of 3 or higher on 56.8% of the days evaluated. The grouptreated with 4.0 μg/dose anti TNF antibody exhibited scores of 3 orhigher on 46.4% of the days evaluated. The 11.9% reduction seen in thehigh dose group was highly significant when compared with thevehicle-treated group (p<0.025).

In this study, the final day of dosing with antibody was on day 12. Atthat time, only 5 of the 28 animals in the study had developed ulcers(grade 3 mucositis). In the high dose antibody group, only 1 animal hada score of 3 on day 12, and the majority of animals (5/8) had a score ofless than 2. The efficacy of antibody treatment under these conditionsdemonstrate that topically applied antibody accessed the area beneaththe mucosal barrier, even prior to the formation of frank ulcers.

Example 2 Trafficking of Topically Applied Antibody in Irradiated OralMucosa

Mucositis is induced in Syrian Golden hamsters (male, 5-6 weeks old, 5animals per group, Charles River Laboratories) using a standardizedacute radiation protocol. A single dose of radiation (40 Gy/dose) isadministered to all animals on day 0. Radiation is generated with a 160kilovolt potential (15-ma) source at a focal distance of 50 cm, hardenedwith a 0.35 mm Cu filtration system. Irradiation targets the left buccalpouch mucosa at a rate of 2.0 Gy/minute. Prior to irradiation, animalsare anesthetized with an i.p. injection of Ketamine (160 mg/ml) andXylazine (8 mg/ml). The left buccal pouch is everted, fixed and isolatedusing a lead shield.

At varying times after radiation, anti-TNF antibody isolated from thecolostrum of cows immunized with murine TNF is applied to both the leftand right cheek pouches and animals are sacrificed 1 hr later. Two timepoints are examined: day 6, when mucositis is beginning to develop, andday 14, when the severity of mucositis is at its peak.

The location of bovine antibody is tracked using polyclonal sheepanti-bovine IgG (h+1) antibody. Sections of the buccal mucosa are fixedin 10% buffered formalin and embedded in paraffin using standardtechniques and longitudinal sections (5 μm thick) are cut. Slides aredeparaffinated, rehydrated and treated with ExtrAvidin (Sigma-Aldrich)to inhibit endogenous biotin background. Slides are blocked with 1%gelatin in PBS and incubated with biotin labeled sheep anti-bovine IgG(h+1). After washing, the slides are treated with alkalinephosphatase-labeled ExtrAvidin, followed by BCIP/NBT as a substrate. Thelocation of bovine immunoglobulin is determined microscopically. Slidesare read by 2 individuals blinded as to the identity of the samples.Five slides are prepared and read from each cheek pouch and scored on ascale of 0-3 (0, no detectable antibody; 1, low level antibody staining;2, moderate antibody staining; 3, intense antibody staining). Little orno antibody is detected in the control, non-irradiated cheek. Anti-TNFantibody is seen penetrating the buccal mucosa in the irradiated cheekpouch at days 6 and 14.

Example 3 Treatment of GI Mucositis in Chemotherapy-Induced MucositisModel

GI mucositis in mice is induced by intraperitoneal administration ofirinotecan hydrochloride (75 mg/kg) or saline to male Swiss mice (25-35g) once per day for 4 consecutive days. Starting on day 1, mice areadministered immunoglobulin purified from the colostrum of cowsimmunized with murine TNF; immunoglobulin is administered by oral gavagetwice per day. Mice receive anti-TNF antibody or control anti-influenzaantibody (20 mg per dose). The animals are evaluated daily through day 7for the presence of diarrhea. Diarrhea observed after the final dose ofirinotecan is considered to be delayed onset diarrhea. The severity ofdiarrhea is assessed using the following scale: 0—normal, normal stoolor absent; 1—slight, slightly wet and soft stool; 2—moderate, wet andunformed stool with moderate perianal staining of the coat; and3—severe, watery stool with severe perianal staining of the coat. Dosingwith anti-TNF antibody, but not anti-influenza antibody reduces theincidence of severe delayed onset diarrhea.

Example 4 Treatment of Burn-Induced Intestinal Injury with Oral Anti-TNFAntibody

Severe burn induces intestinal injury and apoptosis of the intestinalepithelium. Male C57BL/6 mice (25-30 g, 5 mice per group) areanesthetized with methoxyflurane by inhalation and buprenorphinehydrochloride (0.1 mg/kg) by subcutaneous injection. The dorsum of thetrunk is shaved and exposed to a steam burn in a 3×4 cm section of theback, resulting in a 30% surface area scald burn. Sham control animalsare anesthetized, shaved and handled identically to the burned animalswithout exposure to steam. Animals are resuscitated with 0.9% NaCl—1 mls.c. and 1 ml i.p. Immediately prior to administration of the burn (orsham burn), mice are administered a single dose of immunoglobulin byoral gavage. Immunoglobulin is purified from the colostrum of cowsimmunized with murine TNF or control anti-influenza antibody (20 mg perdose). Animals are sacrificed 12 hr after injury and the small intestineis excised, flushed with saline and weighed. A 2 cm section of theproximal small bowel is fixed in 10% formalin. Formalin fixed tissuesare embedded in paraffin and three 3 μm sections are obtained at 40 μmintervals, deparaffinized, rehydrated and washed. H&E staining isperformed and mucosal height, crypt depth and villus height isdetermined by measuring 10 randomly selected villi from each section.Injured animals are found to have reduced small bowel weight, reducedmucosal height and reduced villus height when compared to sham burnedanimals. Treatment with anti-TNF antibody, but not controlanti-influenza antibody, minimizes these pathological burn-inducedchanges.

Example 5 Trafficking of Topically Applied Antibody in the SmallIntestine in Mice Suffering Severe Burns

Severe burn is known to induce intestinal injury and apoptosis of theintestinal epithelium. Male C57BL/6 mice (25-30 g, 5 mice per group) areanesthetized with methoxyflurane by inhalation and buprenorphinehydrochloride (0.1 mg/kg) by subcutaneous injection. The dorsum of thetrunk is shaved and exposed to a steam burn in a 3×4 cm section of theback, resulting in a 30% surface area scald burn. Sham control animalsare anesthetized, shaved and handled identically to the burned animalswithout exposure to steam. Animals are resuscitated with 0.9% NaCl—1 mls.c. and 1 ml i.p. Immediately prior to administration of the burn (orsham burn), mice are administered a single dose of immunoglobulin byoral gavage. Immunoglobulin is purified from the colostrum of cowsimmunized with murine TNF or control anti-influenza antibody (20 mg perdose). Animals are sacrificed 12 hr after injury and the small intestineis excised and weighed. 3 cm sections from the proximal and distal smallintestine are fixed in paraffin and sectioned. Slides aredeparaffinated, rehydrated and treated with ExtrAvidin (Sigma-Aldrich)to inhibit endogenous biotin background. Slides are blocked with 1%gelatin in PBS and incubated with biotin labeled sheep anti-bovine IgG(h+1). After washing, the slides are treated with alkalinephosphatase-labeled ExtrAvidin, followed by BCIP/NBT as a substrate. Thelocation of bovine immunoglobulin is determined microscopically. Fiveslides from each location will be prepared and read from each animal andscored on a scale of 0-3 (0, no detectable antibody; 1, low levelantibody staining; 2, moderate antibody staining; 3, intense antibodystaining Anti-TNF antibody is detected below the mucosal barrier inburned mice, but not in sham-burned mice.

Example 6 Effect of Glycosylation on Stability of Bovine Immunoglobulinto Gastric Digestion

The stability of bovine immunoglobulin to GI digestion is assessed invitro. Bovine immunoglobulin is purified from colostrum. Bovinecolostrums are collected from 6 cows on days 1-4 after parturition,pooled and frozen at −20° C. until further use. Colostrum is thawed andcentrifuged at 4000×g to remove fat. The pH is adjusted to 4.6 using 1 NHCl, incubated for 30 min at 37° C. to precipitate casein, andcentrifuged. The pH is raised to 7.2 and the whey is stored at −20° C.Immunoglobulin is purified by precipitation with 50% (vol/vol) saturatedammonium sulfate and ammonium sulfate is removed by diafiltration. TheIg-enriched colostral whey is fractionated on a column of immobilizedjacalin (Pierce, Rockford, Ill.); jacalin is a lectin that recognizes anO-linked glycan present on human IgA and a subset of bovine IgG. Theimmunoglobulin solution (10 mg/ml) is loaded onto the column and washedwith PBS. The flow-through material (jacalin-non-binding) is collectedand the column is extensively washed. Bound material is eluted with 0.4M D-galactose (jacalin-binding) and dialyzed against PBS.

To mimic transit through the stomach, purified IgG is incubated forvarying times with pepsin, pH 2.0 at 37° C. IgG is buffer exchanged into0.2 M acetate, pH 2.0. Five ml samples at 1.5 mg/ml are incubated at 37°C. with or without 0.05 mg/ml pepsin (Sigma-Aldrich, St. Louis, Mo.).One ml aliquots are removed at varying times (1, 2, 4, 6 hr), and the pHraised by adding 200 μl Tris base. Samples are dialyzed into PBS andstored at 4° C. with 0.02% NaN₃. Samples are analyzed by SDS-PAGE todetermine the degree of immunoglobulin fragmentation. Jacalin-bindingimmunoglobulin is found to be more stable to digestion than isjacalin-non-binding immunoglobulin.

Example 7 Effect of Glycosylation on Stability of Bovine Immunoglobulinto Digestion with Bacterial Proteases of the Oral Cavity

Bovine immunoglobulin is purified from colostrum. Bovine colostrums arecollected from 6 cows on days 1-4 after parturition, pooled and frozenat −20° C. until further use. Colostrum is thawed and centrifuged at4000×g to remove fat. The pH is adjusted to 4.6 using 1 N HCl, incubatedfor 30 min at 37° C. to precipitate casein, and centrifuged. The pH israised to 7.2 and the whey is stored at −20° C. Immunoglobulin ispurified by precipitation with 50% (vol/vol) saturated ammonium sulfateand ammonium sulfate is removed by diafiltration. The Ig-enrichedcolostral whey is fractionated on a column of immobilized jacalin(Pierce, Rockford, Ill.); jacalin is a lectin that recognizes anO-linked glycan present on human IgA and a subset of bovine IgG. Theimmunoglobulin solution (10 mg/ml) is loaded onto the column and washedwith PBS. The flow-through material (jacalin-non-binding) is collectedand the columns are extensively washed. Bound material is eluted with0.4 M D-galactose and dialyzed against PBS (jacalin-binding).

Bacterial strains isolated from the oral cavity are obtained fromAmerican Type Culture Collection (Rockville, Md.). Ten strains areexamined: Capnocytophaga ochracea (ATCC#27872), Streptococcus mutans(ATCC#700611), Streptococcus intermedius (ATCC#31412), Prevotellaintermedia (ATCC#15032), Prevotella intermedia (ATCC#15033), Prevotellanigrescens (ATCC#25261), Prevotella loescheii (ATCC#15930),Prophyromanas catoniae (ATCC#51270), Treponema denticola (ATCC#700771)and Lactobacillus plantarum (ATCC#BAA-793). All strains are cultivatedunder the conditions recommended by ATCC. Individual colonies from 3- to4-day plate cultures are suspended in 40 μl of a 5 mg/ml solution ofpurified immunoglobulin in 0.85% NaCl 0.05 M Tris-HCl, pH 7.4. Purifiedhuman IgG and IgA (SigmaAldrich) are used as controls. After 18 hrincubation at 35° C., samples are analyzed by SDS-PAGE under reducingconditions. Some bacterial strains are found to express proteases thatdegrade human IgA and IgG. At least some proteases are found to haveless activity against bovine immunoglobulin than against humanimmunoglobulin. At least some proteases are found to have less activityagainst jacalin-binding bovine immunoglobulin than againstjacalin-non-binding bovine immunoglobulin.

Example 8 Variability of Glycosylation in Bovine ColostralImmunoglobulin

Colostrum is collected on the day following calving from 12 immunizedcows (six cows are immunized with murine TNF and six are immunized withgliadin) and frozen at −20° C. until further use. Samples from each coware handled separately. Colostrum is thawed and centrifuged at 4000×g toremove fat. The pH is adjusted to 4.6 using 1 N HCl, incubated for 30min at 37° C. to precipitate casein, and centrifuged. The pH is raisedto 7.2 and the whey is stored at −20° C. Immunoglobulin is purified byprecipitation with 50% (vol/vol) saturated ammonium sulfate and ammoniumsulfate is removed by diafiltration. Samples from each colostrum sampleare separated on SDS-PAGE and Western blotted with biotinylated jacalin(Vector Laboratories, Burlingame, Calif.). Colostrum samples fromdifferent animals are found to display different levels of jacalinbinding.

Example 9 Effect of Pregnancy on Antibody Glycosylation

A serum sample is collected from a pregnant cow within one week of theexpected parturition date and a second sample is collected approximatelyone day after calving. Serum samples are also collected from anever-pregnant female cow and an age-matched steer. Glycosylation ofantibodies is detected by ELISA. ELISA plates are coated with rabbitanti-bovine (IgG+IgA+IgM) antibody (Bethyl Laboratories, Montgomery,Tex.) and washed. Two-fold serial dilutions of each serum sample areapplied to the microtiter plates in triplicate wells and washed again.Plates are developed using biotin-labeled jacalin (Vector Laboratories,Burlingame, Calif.) followed by HRP-labeled streptavidin and OPD. Serumimmunoglobulin from a cow near the time of birth is found to display adifferent level of jacalin binding than is immunoglobulin from a male ornever-pregnant female cow.

Example 10 Measurement of Glycosylation of Bovine Immunoglobulin forProcess Development and Release Testing

Fifty cows are immunized with gliadin to generate antibody to be used inthe treatment of celiac disease. Day one colostrum samples from each coware assayed for jacalin binding as a measure of O-glycosylation.Colostral whey is prepared from each sample and assayed by ELISA onplates coated with rabbit anti-bovine IgG1 and developed withbiotinylated jacalin and HRP-streptavidin. Colostrum samples that haveglycosylation levels that fall within pre-defined specifications areaccepted and used for manufacture of the commercial antibody product.Colostrum samples that fall outside of the pre-defined specificationsare rejected and the colostrum is discarded.

Example 11 Effect of Glycosylation on Ability of Antibody to PenetrateDamaged Mucosal Membranes

Anti-TNF antibody is isolated from the colostrum of cows immunized withmurine TNF. The antibody is fractionated and processed to generateseparate preparations with defined patterns of glycosylation. Fourpreparations are compared: 1) jacalin-non-binding antibody, prepared bypassing the antibody over a jacalin-agarose column; 2) jacalin-bindingantibody, prepared by binding the antibody to a jacalin-agarose columnand eluting the antibody with 0.4 M D-galactose; 3) a glycoform ofantibody in which sialic acid and terminal β-galactose residues areremoved by incubation of antibody (100 mg in 10.0 ml in 100 mM MESbuffer, H 7.0) with sialidase A (A. ureafaciens, 100 milliunits) andβ-galactosidase (D. pneumoniae, 100 milliunits) for 24 hr at 37° C.; 4)a glycoform of antibody which is maximally sialylated by incubation ofantibody (100 mg in 10.0 ml in 100 mM MES buffer, pH 7.0) with 50milliunits each of β1,4-galactosyltransferase andα-2,3-sialyltransferase and 5 umol each of UDP-galactose,CMP-N-acetylneuraminic acid and MnCl₂ for 24 hr at 37° C.

Mucositis is induced in Syrian Golden hamsters (male, 5-6 weeks old, 5animals per group, Charles River Laboratories) using a standardizedacute radiation protocol. A single dose of radiation (40 Gy/dose) isadministered to all animals on day 0. Radiation is generated with a 160kilovolt potential (15-ma) source at a focal distance of 50 cm, hardenedwith a 0.35 mm Cu filtration system. Irradiation targets the left buccalpouch mucosa at a rate of 2.0 Gy/minute. Prior to irradiation, animalsare anesthetized with an i.p. injection of Ketamine (160 mg/ml) andXylazine (8 mg/ml). The left buccal pouch is everted, fixed and isolatedusing a lead shield.

After 8 days, when the buccal membrane is partially permeabilized,antibody is applied to both the left and right cheek pouches and animalsare sacrificed 1 hr later. The location of bovine antibody is trackedusing polyclonal sheep anti-bovine IgG (h+1) antibody. Sections of thebuccal mucosa are fixed in 10% buffered formalin and embedded inparaffin using standard techniques and longitudinal sections (5 μmthick) are cut. Slides are deparaffinated, rehydrated and treated withExtrAvidin (Sigma-Aldrich) to inhibit endogenous biotin background.Slides are blocked with 1% gelatin in PBS and incubated with biotinlabeled sheep anti-bovine IgG (h+1). After washing, the slides aretreated with alkaline phosphatase-labeled ExtrAvidin, followed byBCIP/NBT as a substrate. The location of bovine immunoglobulin isdetermined microscopically. Slides are read by 2 individuals blinded asto the identity of the samples. Five slides are prepared and read fromeach cheek pouch and scored on a scale of 0-3 (0, no detectableantibody; 1, low level antibody staining; 2, moderate antibody staining;3, intense antibody staining). The extent of antibody crossing thedamaged mucosal membrane is found to be dependent on the glycosylationstate of the antibody.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished. United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. All other published references, documents,manuscripts and scientific literature cited herein are herebyincorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims. It should also be understood thatthe embodiments described herein are not mutually exclusive and thatfeatures from the various embodiments may be combined in whole or inpart in accordance with the invention.

1. A stabilized antibody for topical administration to the digestivetract.
 2. The antibody of claim 1, wherein the antibody is enriched forleast one carbohydrate moiety that confers enhanced stability againstoral or gastric degradation of the antibody.
 3. The antibody of claim 1,wherein the antibody is stabilized by adding at least one carbohydratemoiety that confers enhanced stability against oral or gastricdegradation of the antibody after the antibody has been synthesized. 4.The antibody of claim 1, wherein the antibody has been treated withenzymes to add carbohydrate molecules that confer enhanced stabilityagainst oral or gastric degradation of the antibody.
 5. The antibody ofclaim 1, wherein the antibody has been genetically engineered to expressthe hinge region sequence from bovine IgG1.
 6. The antibody of claim 1,wherein the antibody is a polyclonal antibody.
 7. The antibody of claim1, wherein the antibody is a monoclonal antibody.
 8. The antibody ofclaim 1, wherein the antibody is a fragment of an antibody.
 9. Theantibody of claim 1, wherein the antibody is a polyclonal antibodyderived from milk or colostrum.
 10. The antibody of claim 1, wherein theantibody is a polyclonal antibody isolated from egg yolks.
 11. Theantibody of claim 1, wherein the antibody is a polyclonal antibody thatis enriched for at least one carbohydrate that confers increasedstability to oral or gastric degradation wherein the carbohydrate is anO-linked carbohydrate attached to a Ser or Thr residue in the hingeregion of the antibody.
 12. The antibody of claim 1, wherein theantibody is a polyclonal antibody that has been treated with enzymes toadd at least one carbohydrate that confers increased stability to oralor gastric degradation wherein the carbohydrate is an O-linkedcarbohydrate attached to a Ser or Thr residue in the hinge region of theantibody.
 13. A method of preparing a stabilized antibody of claim 1,comprising the step of fractionating an antibody preparation based onthe expression of at least one carbohydrate moiety that confersincreased stability to oral or gastric degradation using lectin-basedchromatography.
 14. The method of claim 13, wherein at least one lectinused in the lectin-based chromatograph is jacalin. 15-34. (canceled)